In the year 2015-2016, we continued to focus on two major research areas: 1) screening and characterizing drugs for combination therapy and transmission blocking; 2) studying the molecular mechanisms of malaria pathogenesis and molecular signaling using Plasmodium yoelii/mouse model. 1) In collaboration with scientists in National Center for Advancing Translational Sciences (NCATS), we continued to work on two of the derivatives that showed high potency (20 fold) in killing both sexual and asexual parasites. We are making and testing additional derivatives and are working towards bringing the compounds for animal and clinical trials. We also investigated modulation of artemisinin and ion channel blocker interactions by two parasite transporters, PfCRT and PfMDR. We showed positively correlated responses between dihydroartemisinin (DHA) and several channel blockers, suggesting potential shared transport pathways or mode of action. Additionally, we demonstrated that PfCRT and PfMDR1 could also significantly modulate the interactions of the compounds and that the interactions were influenced by the parasite genetic backgrounds. These results provide important information for better understanding of drug resistance and for assessing the overall impact of drug resistance markers on parasite response to artemisinin combination therapies (ACTs). These results were recently published in Scientific Reports (Sci Rep. 2016 May 5;6:25379. doi: 10.1038/srep25379). We recently obtained an US patent for Compounds that treat malaria and prevent malaria transmission (Patent No: US9,375,424 B2; date of patent: June 28, 2016) based on our discovery of Ketotifen in blocking malaria transmission. We are planning for potential clinical trials and testing additional derivatives. 2) Using the rodent malaria parasite Plasmodium yoelii, we have made good progresses in studying parasite-host interaction in several directions: I. Previously, we performed a genome-wide linkage analysis on host responses to infection of progeny from a P. yoelii genetic cross and identified hundreds of parasite genetic loci linked to responses of many host genes. (Cell Rep 2015; 12: 661-672). Based on the analyses, we investigated one of the candidate genes (CD40) and have dissected the molecular mechanisms on how CD40 regulates type I interferon production and affects malaria infection. Data from this work has been submitted for publication (under revision). II. Again, following through another project reported last year (genetic crosses of P. y. negeriensis N67 and P. y. yoelii YM and identification of a candidate gene encoding a putative E3 ubiquitin ligase), we have submitted the data for publication. We are currently revising the manuscript. We are using the CRISPR/cas9 system to disrupt and to modify the E3 ubiquitin ligase gene to further confirm the function of the gene and its role in malaria pathogenesis. III. We are studying the molecular mechanism of inflammatory responses after P. yoelii infection. We are now focusing on pro-inflammatory responses mediated by IFN-gamma and are dissecting the molecular interactions and cell types contributing to inflammation after parasite infection. We are preparing a manuscript for publication currently. IV. We have initiated other projects, including additional host molecules playing a role in type I response during malaria infection. The results will be described in next year's report. V. Additionally, in collaboration with scientists and students at Xiamen University, China, we continued to work on Leucocytozoon, an avian blood parasite. We have developed a PCR-based test to examine parasite transmission and prevalence in southern China. A manuscript from this work has been submitted to PLoS ONE (under revision). In another project, we performed RNA-seg (sequencing of genome-wide RNAs) and investigated introns in 5 and 3 untranslated regions (UTRs). We obtained 56 millions of 100 bp paired-end reads from Plasmodium yoelii nigeriensis NSM with or without mefloquine (MQ) treatment. Comparison of the sequence reads to the YM genome revealed many sequence reads with introns in 5 and 3 UTRs, altered intron/exon boundaries, alternative splicing, overlapping sense-antisense sequences, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM in public databases discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified 84,000 SNPs between the two parasites. The results suggest that the UTR introns play a role in gene expression regulation. We are functionally testing some of the UTR introns in regulating gene expression in vivo. A paper was published from this work (Malar J., 2016, 15:30). In collaboration with Scientists in Sun Yat-sen University in China, we investigated polymorphisms of the artemisinin resistant marker (K13) in Plasmodium falciparum parasite populations of Grande Comore Island 10 years after artemisinin combination therapy. The results were reported in Parasit Vectors (2015 Dec 15;8:634. doi: 10.1186/s13071-015-1253-z).